Corticotropin-releasing hormone directly activates noradrenergic neurons of the locus ceruleus recorded in vitro

Abstract

The neuropeptide corticotropin-releasing hormone (CRH) activates locus ceruleus (LC) neurons, thereby increasing norepinephrine levels throughout the CNS. Despite anatomical and physiological evidence for CRH innervation of the LC, the mechanism of CRH-evoked activation of LC neurons is unknown. Moreover, given the apparent absence of mRNA for CRH receptors in LC neurons, the exact location of action of CRH within the cerulear region is debated. Using in vitro intracellular recordings from rat brainstem, we examined whether CRH exerts a direct effect on LC neurons and which ionic currents are likely affected by CRH. We demonstrate that CRH dose-dependently increases the firing rate of LC neurons through a direct (TTX- and cadmium-insensitive) mechanism by decreasing a potassium conductance. The CRH-evoked activation of LC neurons is, at least in part, mediated by CRH1 receptors and a cAMP-dependent second messenger system. These data provide additional support that CRH functions as an excitatory neurotransmitter in the LC and the hypothesis that dysfunction of the CRH peptidergic and noradrenergic systems observed in patients with mood and anxiety disorders are functionally related.

Figure 1.

Identification and morphology of LC neurons. An LC neuron stained with biocytin (black) after intracellular recording in a 300-μm-thick horizontal slice of the rat brainstem is shown. Immunoreactivity for TH (brown) in somata of surrounding neurons outlines the LC. Immunoreactive processes illustrate the preservation of the extensive arborization of LC neurons outside the nucleus proper, which is where the majority of CRH innervation occurs. Scale bar, 50 μm. IV, Fourth ventricle.

Figure 2.

Bath application of CRH. A, Increasing concentrations of CRH increase the spontaneous discharge rate of LC neurons (maximal increase, 1.5 ± 0.9 Hz evoked by an average concentration of 325 ± 207 nm; n = 4 neurons). The increase in the discharge rate was variable, difficult to quantify, and required an extended period of time to wash out. B, Bath application of CRH increases in the input resistance of LC neurons from an average of 64 ± 5 to 82 ± 10 MΩ at an average concentration of 375 ± 179 nm CRH (n = 4 neurons). C, The response to local administration of AMPA was not reliably altered by bath application of CRH. Measurements for “Washouts 1 and 2” were made between 3 and 7 min and 15 and 30 min after switchover to aCSF, respectively.

Figure 3.

Local administration of CRH increases the FR of spontaneously active LC neurons by increasing the rate of repolarization. A, The average baseline FR of this spontaneously active LC neuron was 0.85 Hz. CRH (105 ng) was ejected (horizontal bar) in the bath ∼400 μm from the recording site. A maximum FR of 2.00 Hz was reached at 12 sec (arrow) after CRH ejection. B, Repeated CRH administration causes an increase in the FR that is transient and dose dependent. The maximal activation in this LC neuron occurs ∼15 sec after CRH ejection, and the FR has returned to baseline levels within 90-120 sec. C, An overlay of five action potential waveforms at the time point of maximal CRH-evoked activation (black) demonstrates that the waveform is similar to the waveform of five action potentials from the same neuron immediately before CRH ejection (gray). At the time of maximal CRH-evoked activation, the rate of repolarization after action potential discharge is faster, leading to a more rapid return to action potential threshold and consequently an increase in the FR of the neuron.

Figure 4.

CRH increases the discharge rate of putative calcium spikes in LC neurons in the presence of TTX. A, When synaptic activity is prevented by bath application of TTX (2 μm), the basal FR of LC neurons decreases significantly. B, This LC neuron continued to discharge calcium spikes spontaneously. C, In the presence of TTX, CRH increased the calcium spike rate in six of six neurons to a similar extent as before TTX. D, Representative trace demonstrating that CRH administration (36 ng; horizontal bar) increased the FR of this neuron under control conditions (aCSF). E, In the same neuron, the calcium spike discharge rate after bath application of TTX (2 μm) was increased from 1.20 to 2.20 Hz at 16 sec after CRH administration.

Figure 5.

CRH increases the FR of LC neurons in the presence of cadmium. A, When calcium-dependent neurotransmitter release is prevented by bath application of the CdCl₂ (100 μm), the basal FR of LC neurons increased significantly. B, This increase occurs most likely as a result of blockade of calcium-activated potassium conductances, leading to a decreased AHP. C, In the presence of cadmium, CRH administration increases the LC FR to a greater extent (0.85 ± 0.21 vs 0.50 ± 0.16 Hz; p < 0.01), but with a similar time course (maximal activation at 15 ± 2 sec) as under control conditions. D, Representative trace demonstrating the CRH-evoked activation in the presence of CdCl₂.

Figure 6.

CRH depolarizes the membrane of LC neurons in the absence of action potential discharge. A, When action potential discharge is prevented by constant hyperpolarizing current delivered through the recording electrode, CRH ejection causes depolarization of the membrane potential. B, The magnitude of the CRH-evoked voltage deflection is voltage dependent with a reversal potential of -114 ± 8 mV (n = 21). C, The magnitude of voltage deflections produced in response to repeated hyperpolarizing current steps (0.3 nA, 10 msec, 1 Hz) increases during the CRH-evoked depolarization, indicating an increase in input resistance. Current-evoked depolarization of the same magnitude as that caused by CRH did not change the input resistance (data not shown).

Figure 7.

The CRH-evoked increase of LC neuron activity was reduced when potassium conductances are blocked by cesium. A, LC neurons recorded with electrodes containing cesium acetate (3 m) exhibit a higher FR. B, Cesium also caused a depolarized membrane potential, a much longer duration action potential, and a reduced AHP. C, Administration of a higher dose of CRH only causes a slight increase in the FR (0.2 Hz). Nevertheless, the FR of the neuron was increased by bath application of glutamate (300 μm).

Figure 8.

The CRH-evoked increase in the FR persists during blockade of several specific potassium conductances. A, Bath application of a blocker of calcium-activated potassium conductances, apamin (200 nm), does not affect the CRH-evoked increase in the LC FR. The effectiveness of apamin blockade on the AHP is evident in an overlay of the action potential waveform obtained before and during apamin administration. B, Bath application of a blocker of inwardly rectifying potassium conductances, BaCl₂ (100 μm), increases the spontaneous FR of LC neurons but does not affect the CRH-evoked increase in the LC FR. The effectiveness of barium in blocking inwardly rectifying potassium conductances is evident from a comparison of the current-voltage plots obtained before and during barium administration.

Figure 9.

The magnitude of the CRH-evoked increase in the FR was reduced by CRH antagonists. A, Local administration of D-Phe-CRH increases the LC FR with a time course similar to the CRH-evoked activation of LC neurons. B, Although bath application of the CRH antagonist D-Phe-CRH (1 μm) also increased the spontaneous FR of LC neurons, it reduced the magnitude of the effect of CRH. Bath application of CP154,526 (1-100 μm) reduced the effect of CRH without increasing the basal FR. The numbers on each bar indicate the number of neurons contributing to the average. C, Vehicle ejection (note the large volume) does not affect the LC FR.

Figure 10.

The increase in the LC neuron FR is prevented by intracellular administration of the PKA inhibitor Rp-cAMP-S but not by H89. A, When tested within the first 5 min of impalement of the neuron with an electrode containing the cyclic nucleotide analog and PKA inhibitor Rp-cAMP-S (4.5 mm; ∼400× IC₅₀ value), CRH administration (50 ng) increased the LC neuron FR. Diffusion of Rp-cAMP-S into the cell decreases the spontaneous FR. Five minutes after the initial activation by CRH, administration of 10-fold higher doses of CRH no longer increases the LC FR. B, In contrast, LC neurons recorded with electrodes containing the PKA inhibitor H89 (10-50 μm; ∼1000× IC₅₀ value) exhibit a response to CRH that is stable over time. C, The increase in the LC FR in response to CRH administration at later time points was expressed as a percentage of the response to CRH immediately after entry of the neuron with electrodes containing Rp-cAMP-S (n = 3) or H89 (n = 4). D, We interpret these data to indicate that CRH activates LC neurons via a cAMP-dependent mechanism independent of PKA.